The aim of this project is to understand how proteins fold within and translocate across membranes by studying the behavior of diphtheria toxin, and to apply the lessons learned to other systems. One of our goals in this project will be to determine the structure of the membrane-inserted toxin at medium-to-high resolution, defining not only what parts of the protein are embedded within the bilayer, but also identifying the secondary structure (which may be a combination of beta-sheets and alpha- helices) and orientation of membrane embedded segments. This will involve systematically mapping out of the depth of individual residues in membrane penetration regions of the protein, and identifying whether individual residues in membrane-embedded segments are exposed to lipid, the interior of the protein, or the aqueous solution. These questions are being tackled by a combination of methods, including immunochemical, biotin/streptavidin, fluorescence and especially a fluorescence quenching method to determine depth recently developed in our laboratory. These approaches will be applied to membrane-inserted toxin and to membrane- inserted mutant toxins in which site-directed mutagenesis and chemical labeling are combined to label single residues with fluorescent groups or biotin. The translocation of the toxin across membranes is also being studied. We are using an in vitro translocation system that allows control of translocation conditions to characterize the translocation process and pore formation by the toxin. In additional studies, we will begin to look at the mechanism of membrane translocation of ordinary cellular proteins through studies of the SecA protein. We have already found that SecA, which plays a central role in E. coli translocation, shows significant parallels in its behavior to that of diphtheria toxin. These studies will contribute greatly to our understanding of membrane protein insertion and structure, protein translocation and the behavior of bacterial toxin proteins. The new methods we are developing should be applicable to the analysis of membrane protein structure at medium-to-high resolution in many systems. In addition, these studies should have a significant impact on the design of therapeutically useful "immunotoxin" agents, an area of intense applied biomedical research, and in the development of more effective vaccines and therapeutic agents for bacterial diseases where toxin proteins are important virulence factors.